Monoclonal antibody-based enzyme immunoassay for Giardia lamblia antigen in human stool

Abstract

A visually readable monoclonal antibody-based antigen-capture enzyme immunoassay for the detection of Giardia lamblia antigen in human stool specimens was developed and found to be 97% (30 of 31 stool specimens) sensitive for formalinized stools and 82% (49 of 60 stool specimens) sensitive for unfixed stool specimens by visual reading. The storage of specimens in 10% Formalin resulted in increased absorbance in 20 of 26 G. lamblia-positive specimens tested as both formalinized and unfixed specimens; the increase averaged 1,336%. The assay was specific for antigens of this organism and for antigens derived from the cyst, as opposed to the trophozoite, stage. The assay could detect the antigens of five cysts per well, but could not detect antigen in in vitro-cultured trophozoites. A mouse monoclonal antibody of the immunoglobulin G1 (IgG1) subclass, which was prepared against cysts of G. lamblia, was used as the solid-phase capture antibody. The antibody was reactive with the cyst wall, as determined by immunofluorescence. Polyclonal rabbit anti-cyst IgG was used as the secondary antibody, and peroxidase-labeled goat anti-rabbit IgG was used as the tertiary antibody in the assay format. Maximal capture of antigen from stool specimens occurred by 30 min. Optimal dilution of specimens was in the range of 1:60 to 1:600. Preliminary characterization of affinity-purified antigen recognized by the monoclonal antibody showed that it is heat stable (100 degrees C, 12 min) and resistant to sodium periodate treatment and that it may exist in multiple molecular weights from 45,000 to 110,000.